Alcoholism is a major health concern in the VA population as well as in the general population. An integral part of the mission of the VA is to provide care for VA patients suffering from alcohol and/or drug abuse problems. Alcohol tolerance plays a major role in both the development of alcohol dependence and in promoting alcohol intake. It is also known that alcohol can produce anxiolytic effects in both human and animal models and that rapid tolerance to the anxiolytic responses of acute ethanol also develops. Thus, alcohol tolerance can thereby promote increased alcohol drinking and maintain alcohol addictive behaviors. Amygdaloid brain structures are known to play an important role in anxiety behaviors. It has been show that rapid tolerance elicits neuroadaptation and is also a good predictor of chronic tolerance. The molecular mechanisms in specific neural circuitries of the brain that are involved in the development of rapid tolerance to the anxiolytic effects of ethanol are not well understood. Brain-derived neurotrophic factor (BDNF) plays a role in neuronal survival, differentiation, and consolidation of synaptic strength. Binding of BDNF to tyrosine kinase B receptors triggers the activation of the mitogen-activated protein (MAP) kinase signaling pathway, which phosphorylate of cAMP response element binding (CREB) protein, leading to the regulation of neuronal function. Recently, it has been shown that BDNF can regulate both dendritic spine morphology and function by modulating the function of microRNAs (miRNAs) and its targets, p250GAP, a GTPase activating protein and Limk1 proteins in the brain. miRNAs are small non-coding RNAs that have been shown to regulate gene expression at the post- translational level. Two miRNAs (miRNA132, and 134) in particular in the brain, may potentially be regulated by BDNF, resulting in the regulation of dendritic spine morphology and function. It appears that BDNF may regulate spine function via three potential cellular mechanisms. First, miRNA132, which is a CREB target gene, may regulate dendritic spine function by controlling the expression of p250GAP. Second, miRNA134, whose expression is regulated by BDNF, controls the expression of an mRNA encoding a protein kinase, Limk1, which has been shown to regulate spine development. Third, the effector immediate-early gene Arc (activity- regulated cytoskeleton associated protein) may be relevant in regulating dendritic spine density (DSD) because it is rapidly induced by synaptic stimulation and is also localized to both dendrites and spines. Arc is also a CREB target gene and is potentially induced by BDNF. Importantly, none of the above mentioned molecular mechanisms have been investigated in the process of alcohol tolerance. The specific hypotheses are: 1) rapid tolerance to the anxiolytic effects of acute ethanol may be due to cellular tolerance at the level of BDNF expression and related signaling pathways, thereby affecting dendritic spines in the central amygdala (CeA) and medial amygdala (MeA), but not in the basolateral amygdala (BLA); and 2) tolerance at the level of BDNF, via regulation of the expression of miRNAs 132 or 134, or Arc, may be involved in regulating dendritic spines in the CeA or MeA, thereby playing an operative role in the molecular mechanisms of alcohol tolerance. The immediate goals of the proposed studies are to examine: a) the role of BDNF and its related target genes, including Arc and miRNAs 132 and 134, in rapid tolerance to the anxiolytic responses of ethanol using a rat model; b) if the reversal of alcohol tolerance by BDNF is mediated via miRNA132, miRNA134, or Arc in the CeA and MeA; and c) if BDNF regulates the development of rapid tolerance via regulation of miRNAs or Arc- mediated regulation of dendritic spines in the CeA and MeA but not in BLA of rats. The proposed studies will provide novel information on the molecular mechanisms in the neurocircuitry of the amygdala that may be involved in alcohol tolerance and will facilitate the identification of molecular targets, including miRNAs and related signaling pathways, that can be used in developing new pharmacotherapies for both alcoholism and related co-morbid anxiety disorders. PUBLIC HEALTH RELEVANCE: Public Health Relevance: Alcohol abuse and anxiety disorders are major health concerns in the VA population. Veterans suffering from anxiety disorders alone or in combination with post traumatic stress disorder are more prone to developing alcoholism and drug addiction. This proposal will enhance our understanding of the molecular mechanisms associated with the development of alcohol tolerance and anxiety- like behaviors and will identify molecular targets that can be used to develop future therapeutic agents in treating alcohol-abuse disorders with and without anxiety disorders.